The present invention relates to a new separation method and a device therefor. The invention is especially related to a method and a device for the separation of different components or species in a given sample for the purpose of their identification, wherein each different species has a specific and different residence time in a separation channel or is irreversibly retained at a specific and different position in said channel.
In chromatographic chemical analysis methods, a sample containing one or more unknown components (sample species) is contacted with a carrier fluid (usually referred to as the mobile phase) which carries the sample solutes through a separation channel (or column) in which a retentive layer (usually referred to as the stationary phase) is arranged. During their motion through the separation channel, the sample species are continuously exchanged between the retentive layer and the mobile phase (the mobile phase is usually selected such that it has no or only a small affinity for the retentive phase). As the different sample species have a different affinity for the retentive layer, one species will spent more time in the retentive layer than another. As a consequence, all different species will move through the separation-channel with a different velocity. Arranging a detector device at the end of the separation channel, the different sample species will pass the detection point in a clearly separated mode. The response signal which is obtained when each ensemble of identical sample species passes the detector is commonly referred to as a peak.
To obtain an optimal performance, the width (i.e., in the time domain) of this peak should be as small as possible compared to the mean channel residence time of the given ensemble of sample species.
Presently, the two most popular chromatographic techniques are either pressure-driven or electrically driven. In pressure-driven chromatography, the mobile phase motion is generated by applying a pressure difference across the separation channel. The two most popular versions of pressure-driven chromatography are packed-column liquid chromatography (HPLC) and open-tubular gas chromatography (capillary GC), which are characterized in that different sample species have a different and unique residence time in the separation channel, and which are also characterized in that all sample species are detected only once. These two chromatographic techniques suffer from the fact that the pressure drop may not exceed a given value. As can be learned from Poiseuille""s law, this pressure-drop limitation restricts the allowable column length and the applicable mobile phase velocity. Poiseuille""s law also shows that the pressure drop limitation also puts a down limit on the effective column diameter d (open-tubular columns) or particle diameter dp (packed columns) which can be used when a given separation quality has to be achieved. The latter restriction puts a second down-limit on the analysis time, because the analysis time can be considered to be proportional to d2 or dP2. In electrically driven separations, a similar down-limit on the analysis time exists. In this case, the down-limit originates from the existence of a maximal allowable voltage drop.
The documents U.S. Pat. No. 4,874,507 and DE 41 08 820 describe continuous separation devices in which two oppositely moving surfaces are used to transport electrically charged or absorbed solid particles or fluid substances in two different directions. These devices and methods only enable to divide a liquid or solid feed stream into two different fractions. A complete single step fractionation is impossible, nor is it possible to characterize the nature of the different sample components from their unique and different residence time in the channel, as is possible with the methods known to those skilled in the art of HPLC or capillary GC.
In patent application EP 0 670 489 (Manz, 1997) a closed annular separating device is presented in which the use of a freely rotating internal toroidal ring is proposed as one of the possibilities to recirculate (i.e., without creating any net fluid displacement) the mobile phase in a closed channel.
The fact that a closed (annular) envelope is needed to cope with the pressure build-up inside the device restricts the field of application to batch mode separations (i.e., no inflow of fresh eluting mobile phase fluid during the actual separation). The device and method in EP 0 670 489 is hence not related to the type of once-through analytical separations known to those skilled in the art of HPLC or capillary GC. The device and method in EP 0 670 489 generates a complex system of continuously revolving and overtaking substance peaks, potentially causing undesirable competitive adsorption effects when two already separated substance peaks overtake each other. Furthermore, when the sample contains a large number of different species, inextricable chromatograms are obtained. In contrast thereto, it is a main objective of the present invention to provide a pressure-drop less operation of the type once-through analytical separation methods known to those skilled in the art of HPLC or capillary GC, characterized in that different sample species have a different and unique residence time in the separation channel, and which are also characterized in that all sample species are detected only once.
In the method according to the present invention, the separation occurs in a separation channel, said channel being defined by at least two channel elements, and said channel being substantially sealed along its mantle surface, characterized in that the movement of the mobile phase in, through and out the separation channel is mainly caused by a relative axial movement of at least one of the channel elements compared to at least one of the other channel elements. In the present text, the notion xe2x80x9cmoving channel elementxe2x80x9d is used to refer either to a movable part of the channel wall or to a movable mechanical device positioned in the channel""s interior.
In the device according to the invention the thickness of the channel is between 0.01 micron and 100 micron and preferably between 0.1 and 10 micron, and the width of the channel is between 0.1 micron and 10 centimeter and preferably between 10 and 1000 micron.
The methods according to the present invention are based upon the fact that, instead of applying a (pressure) force at the channel inlet only, the motion of the mobile phase fluid is generated by applying a force all along the column length. As a consequence, the mobile phase flow is generated without creating a pressure drop.
In the embodiments according to the present invention, the desired mobile phase flow is at least partly generated by the shear forces which originate from this moving element. In some of the embodiments according to the present invention, the mobile phase flow is furthermore sustained by one or more relief elements, such as one or more protrusions, recesses, holes or irregular porous-like structures, which are provided on the surface of the moving elements.
This implies that in all the embodiments according to the present invention, the mobile phase motion is generated without creating a pressure drop; and hence without the need to impose an excess pressure at the channel inlet, which pressure differences are considered as a basis for potential separation and identification problems. This explains why the moving channel element can also be apart of the channel wall, because, as the pressure inside the channel is substantially identical along its entire length, this pressure can be kept substantially equal to the pressure outside the channel such that the sealing of a channel mantle which consists of two independently movable wall elements poses no specific problem.
To obtain a suitable operation of the method and the device according to the present invention, any specific affinity between the surface of the moving channel elements determining the direction of the mobile phase flow and any of the mobile phase and sample fluid components should be excluded.
As will be shown below, the possibility to perform a chromatographic chemical analysis without creating a pressure drop offers a large number of advantages compared to the conventional pressure-driven chromatography. As already mentioned, the quality of the separation between two different sample species is maximal for a minimal ratio of the width of the eluted peaks to their residence time in the separation channel.
It can be learned from chromatographic theory that this ratio increases with the channel length. Considering now that the method according to the present invention puts no limit on the channel length, a first important advantage compared to the pressure-driven chromatography is noted.
Other advantages (e.g., larger efficiency per unit column length, faster separation times) originate from the fact that the method according to the present invention also allows to limit the following peak broadening phenomena typically encountered in chromatographic separations:
the finite time needed for the mass transfer in the mobile phase
i) the finite time needed for the mass transfer in the retentive phase the presence of lateral variations of the mobile phase velocity or of the ratio of mobile to retentive phase thickness when using channels with a large aspect ratio cross-section the molecular diffusion and longitudinal dispersion in the mobile phase
ii) the velocity gradient (s) in the mobile phase (y and z-direction).
As can be learned from chromatographic theory, the contribution of the phenomena i) and ii) to the peak broadening can be minimized by minimizing the channel diameter (open-tubular channels) or the particle diameter (packed channels). Whereas in pressure-driven chromatography a minimal channel or particle diameter is imposed via Poiseuille""s law, the channel diameters which can be used according to the presently proposed method are only restricted by practical manufacturing and detection limits.
When considering columns with a circular or circular-like cross-section, the use of small (i.e., sub micron) diameter columns brings about major detection problems. Using mass flow sensitive detectors (such as e.g., a mass spectrometer), the detection suffers from the extremely small mass flow rates. Using on-column optical detection methods, the detection suffers from the extremely short optical path lengths. For these reasons, a preferred embodiment for the method according to the present invention involves the use of separation channels with a flat rectangular cross-section, allowing to combine the fast separation kinetics resulting from the small channel thickness with the large optical path length and flow rate resulting from the large column width. Preferentially, the thickness of the channel should be between 0.01 micron and 100 micron, and more specifically between 0.1 and 10 micron, and the width of the channel should preferentially be between 0.1 micron and 10 centimeter, more specifically between 10 micron and 1000 micron. As indicated under point iii) of the above list, the use of separation channels with a flat rectangular cross-section however might cause some undesired peak broadening effects. In the present text, a number of solutions (specially adapted manufacturing methods and/or the use of specific guiding means) is given to minimize the lateral variations of the mobile phase velocity or of the ratio of mobile to retentive phase thickness. An even more preferred proposed method is based upon the use of moving channel elements which allow to accommodate the entire mobile phase fluid into a plurality of compartments, said compartments being arranged such that the direct exchange of fluid elements between said compartments is substantially prevented.
One of the advantages of the use of such a compartmentalized embodiment is that the fluid near the side walls is not retarded with respect to the flow in the central portion of the channel. This implies that lateral variations (z-direction) of the mobile phase velocity are excluded. This is a feature which cannot be obtained with any presently existing chromatographic apparatus. Furthermore, the compartmentalized embodiment allows to add means which promote the mixing in the mobile phase such that the effect of lateral variations of the capacity ratio (i.e., the ratio of mobile to retentive phase thickness.) is eliminated by continuously redistributing the sample solutes in the lateral direction, while, due to the presence of the compartment barriers, these increased mixing rates do not cause any significant peak broadening. The possibility to limit the effect of the molecular diffusion and/or the longitudinal dispersion (see iv) to a single compartment is another feature which cannot be obtained with any presently existing chromatographic apparatus. Another advantage of a compartmentalized flow system can be found in the fact that in a shear-driven open-channel flow, variations in channel depth or channel width induce flow width induce flow mal-distributions, stagnant fluid layers and undesired velocity gradients. These effects may cause an unallowably large peak broadening. By organizing the flow into non-intermixing compartments, a fixed flow rate is obtained, independent of the variations in the cross-sectional dimensions. The manufacturing tolerances for a compartmentalized mobile phase flow are hence less stringent than for an open-channel shear flow. Another advantage of the compartmentalized flow is related to point v) of the above list, because all the fluid elements contained in a given compartment move trough the channel with the same net axial velocity such that there is substantially no net velocity gradient in the direction of the channel thickness (y-direction). The absence of such a velocity gradient is also a feature which cannot be obtained with any presently existing chromatographic apparatus.
In the methods according to the present invention, the channel may have any possible longitudinal shape, including straight, circular, spiral, helicoidal shapes. When having suitable thermal expansion characteristics, the channel elements can be made from any possible material (metal, semi-conductor, polymer, glass-like . . . ) or combinations of materials. If required, part of the surface of the elements can be coated with an inert, wear-free layer. To obtain a sufficient flexibility for the moving elements, segmented and/or laminated elements can be considered.
The devices according to the present invention can be operated at elevated temperatures and the column pressure (which is substantially identical at each point along the channel) can be put at any desired value (i.e., atmospheric and supra- and even sub-atmospheric). The device can hence be used to perform gas, liquid and super-critical fluid chromatography.
In a very attractive embodiment (the so-called Opposite-Moving-Channel-Elements device), the retentive phase is also subjected to a relative motion (opposite to the movement of the inert column parts) with respect to the detection point, while keeping the detection point fixed in space. In this way, the separation quality which can be achieved in a given column length can be drastically increased.
In the methods according to the present invention, the separation occurs basically in an open-tubular channel with freely accessible in- and outlet ports. This implies that the action of the moving channel elements can easily be combined with any other type of force field (electrical field, pressure force, gravitational, centrifugal force). This additional force field can for example be used to sustain the mobile phase flow, or to transport the fluid phase from the sample and mobile phase pre-treatment section to the separation channel and/or from the separation channel to the detection section, or even to create an additional separation effect.